The structure and function of bacterial protein toxins were analyzed using biochemical, genetic, and cell biology methods. Our previous work showed that the anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of sensitive cells, is cleaved by the cell surface protease furin, and then captures either of the two other proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis and reach an acidic compartment from which LF and EF escape to the cytosol. EF is a calcium- and calmodulin-dependent adenylyl cyclase which causes large and unregulated increases in intracellular cAMP concentrations. LF was found to be a metalloprotease that rapidly cleaves mitogen-activated protein kinase kinase 1 (MAPKK1, or MEK1). Cleavage occurs near the N- terminus, after residue 7, and inactivates MEK1. It will now be possible to screen for protease inhibitors that block the action of LF and limit the pathogenesis of anthrax infection. The collaborative study which determined the structure of PA was extended to determine the structure of LF. Large amounts of pure LF obtained by secretion from Bacillus anthracis using a vector in which the promoter and signal peptide of PA are fused to the LF structural gene. Analysis was completed of a large set of mutated PA proteins in which two flexible loops in the receptor-binding domain (domain 4) were randomly substituted by codon mutagenesis with Ala. Substitutions of certain amino acids within the smaller loop greatly decreased binding to receptor. Chemical and retroviral mutagenesis of CHO cells yielded mutants resistant to Clostridium septicum alpha toxin, a hemolysin related to the better-known aerolysin. Analysis showed that the alpha toxin resistant CHO mutants had lost the ability to synthesize glycosylphosphatidylinositol (GPI)-anchors, and the mutation was localized to the second enzyme in the GPI biosynthetic pathway. Comparison of a number of cell lines confirmed that alpha toxin uses (GPI)-anchored proteins as receptors, but the set of proteins is not exactly the same set as used by aerolysin.